Uterine leiomyoma (LM), also known as uterine fibroids, are benign, smooth muscle tumors of the uterus characterized by extensive cellular alteration and stiffness of the extracellular matrix (ECM). Alarmingly, almost 75% of all women will develop some form of fibroids in their lifetimes, with some experiencing significant symptoms. Current medical treatments for LM have off-target side effects that limit their long-term use; surgical options are the only definitive treatment. Consequently, LM is a major gynecologic healthcare problem, and its treatment costs billions of dollars annually. Unfortunately, the molecular mechanisms underlying LM tumorigenesis and progression are poorly understood, and this has posed a significant barrier to the development of new treatment options. The proposed studies are highly significant because they will fill a gap in knowledge about this major gynecologic disease, identifying the mechanisms that drive LM formation as well as potential targets for new LM therapies. Although estrogen and progesterone signaling and TGF? signaling have been implicated in LM, the search for potential driver mutations in LM led to the identification of genes that encode two major proteins that regulate transcription and 3D chromatin topology. Overexpression via chromosomal translocation of the gene encoding chromatin-binding protein high mobility group protein HMGA2 (HMGA2-ra) and mutations in the transcriptional mediator complex subunit Med12 gene (mut-MED12) have been identified as mutually exclusive driver mutations in LM. They together contribute to almost 85% of all LM. Previous cytogenetic, IHC, molecular and whole genome sequencing studies clearly established a role for HMGA2 overexpression in leiomyoma pathogenesis. In this proposal, utilizing these previous observations, we hypothesize that HMGA2 overexpression alters its association with chromatin, thereby changing epigenetic signatures and strongly influencing 3D chromatin topology, which alters gene expression compared to normal myometrial tissues. To test our hypothesis, the following two specific aims will be pursued. Specific Aim 1, we will define the cistrome, transcriptome, and epigenome in HMGA2-ra LM. In Specific Aim 2, we will define chromosomal 3D-topology in HMGA2-ra LM. We will use state-of-the-art genome-wide technology and bioinformatic analysis to achieve these aims. Once completed, the proposed studies will advance our knowledge of this highly prevalent public health challenge in gynecology that affects half of the world population. The proposed work is scientifically, translationally, and clinically significant because these studies will establish a rational pre-clinical framework to assess existing treatments and develop novel therapies for targeting LM, a major gynecologic disease.